JPS59163382A - Novel beta-hydroxy ester and its preparation - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I (X is alpha-CH2, or beta-CH3; Y is alpha-OH, or beta-OH). USE:An intermediate raw material for synthesizing an antibacterial agent. For example, useful as an intermediate raw material for synthesizing erythromycin showing high antibacterial activity against Gram-positive bacteria, useful for microbism, oudemansin showing an inhibitory action of nucleic acid synthesis on Ehrlich ascites carcinoma cell, indolmycin, or applidiasphingosine. PREPARATION:A beta-keto ester shown by the formula II (R1 is 2-tehienyl; R2 is alkyl) is brought into contact with a microorganism belonging to the genus Candida, Endomycopsis, Hansenula, Kloeckera, Lipomyces, Saccaromyces, Schizosaccharamyces, or Sphorobolomyces or its mold, to give the desired compound shown by the formula I .

Description

[Detailed description of the invention] E (wherein, × is α-CH3 or β-C)-
13, Y represents α10H or β-OH. A novel β-hydroxy ester represented by the formula: (
However, in the formula, R represents a coachenyl group, and R2 represents an alkyl group. ) Candlda
) Novel production of hydroxy ester, characterized in that a compound represented by the general formula: (wherein R, R2 are the same as above) is obtained by contacting with various yeasts, including those of the genus It is about law.

Here, as the above β-hydroxy ester, the following '
l 1M optical isomers can be mentioned.

(A) (C) δ)(OH (8) (DJ) (A) and (B) are called Syn bodies, and (C) and (D) are called A.11 bodies.

The target substance of the present invention can serve as an important raw material compound when converted into various useful substances, such as those shown below.

For example, it exhibits strong antibacterial activity against Durham-positive bacteria and has low toxicity, making it useful as an important intermediate raw material in the synthesis of erythromycin, which is widely used for bacterial infections.

It is also useful as an important intermediate raw material when synthesizing the antibiotic Oudemansin, which exhibits strong antibacterial activity and inhibits nucleic acid synthesis against Ehrlitsu ascites-type deer ulcer cells (TlmAn).
Ke et al. The Journal of A
ntlblotlcs. p///2.

Nov. / 9 7 q).

OC83COOCI-13 Furthermore, in the synthesis of indolmycln, which is isolated from Streptomyces albus and has antibacterial properties against certain types of staphylococci that are resistant to various commercially available antibiotics. It is also useful as an intermediate raw material (W, S, Marsh
et at Antlblot.

Chemother, , 10, 3/b (/
(See 9 Otsu 0)).

III-2c,
[Indolmycin] Mθ H Furthermore, it is isolated from Abridium (At) ldlum sp., a type of sea squirt, and exhibits antibacterial and antitumor properties against B and L/210 tumor cells. Cyasphingosine (Apl 1dlasph
It is also useful as an intermediate raw material for l-ngoslne).

In order to synthesize these useful natural products, it is necessary to obtain optically active compounds having the same absolute coordination as the natural products.

As a method for synthesizing these compounds, Mg, Mg+1
A method of aldol condensation of the corresponding erulate and the corresponding aldehyde in the presence of a chelate covalent cation such as HLDA (lithium diisopropylamide) to obtain the corresponding aldol, or using Na8H as a reducing agent.
4. Methods using L I BH4 are known. However, in the former method, the reaction conditions must be set strictly, and the raw material erlate must also be stereochemically regulated. On the other hand, the latter has drawbacks such as insufficient selectivity and the presence of an ester group in the molecule, resulting in excessive reduction.

Moreover, in any case, optically active β-hydroxy esters cannot be obtained by the above-mentioned methods, but only the corresponding racemates are obtained.

Therefore, the present inventors developed the optically active β-
As a result of intensive research into methods for obtaining hydroxyesters, we found that chemical reduction methods were not possible, and we were able to obtain hydroxyesters in high yield and with high optical purity using microorganisms belonging to the glaze genus, including the genus Candida. The present invention was completed based on the discovery that it is possible to obtain an optically active β-hydroxy ester having the following properties.

The present invention will be explained in detail below.

First, β-ketonistyl (IV), which is the starting material of the present invention,
can be obtained, for example, by the following steps.

(IV) (wherein, (I) (in this case, a mixture of the round-shaped isomers (A), (B), (C), and (D) of Group 4a (7)) is obtained [Reformatsky
)reaction〕.

'The given β-hydroxy ester (total) is treated with an oxidizing agent, e.g. Jones reagent, pyridinium dichromate (P
DC)-CH2α2, Cl-13COOH-pyridine-
Treatment with DMSO etc. gives the starting material QV).

In addition, by reacting the obtained β-ketonistyl with the corresponding alcohol, transesterification can be performed to appropriately replace the ester moiety R2 (M, T
an1Huchl et al Chem, Pharm
.

(See Bull9, 2θ, /11.3gC/972).

(IV) (IV') Alternatively, the corresponding ester (R
It can also be obtained by condensation in the presence of DA (lithium-diynzolopyramide) and an alkali metal alcoholade.

The optically active β-hydroxy-ester, which is the target compound of the present invention, can be obtained by using the thus obtained compound (2) as a starting material and contacting it with the following microorganisms or their cells.

, the above microorganism is Candida
Genus, Enclomycopsls
) genus, Hansenula (Hanser+ula) genus, Crockera (Ioeckera) genus, Lipomyces (Ll
pomyces), Saccharomyces (Sacch)
Sc.
hlzosaccharomyces) and Sporo) p.

The microorganism is selected from the group of microorganisms belonging to the genus Sporobolomyces, and representative examples include the microorganisms shown in Table 1.

In Table 1, ゛, &Hiro to 〇7, K/2, and Yakot are microorganisms commissioned by the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 26, 1960, respectively, and V ″FER
MP-" indicates each accession number. All of the above microorganisms are known microorganisms described in the known documents shown below.

CzechOslovak Co11ectlons
of Mlcroorganlsmus.

Catalog of Cultures, Br
no, January /q12'f Nagao Research Institute Fungi Research Report (/?55): The Japan
eseFederatlon of Culture
Co11ectlon ofMlcroorganls
ms, JFCC Catalog of Cult
ures, Ag%A/A, A2 Otsu, and Grave 36 are the preserved strains of the Institute for Fermentation Research (IFO), and are available to anyone [: lN5TITUTE FOR
FERMENTATION O8A, LIST O
F CULTURES/9'7g 4thEDITI
ON: (Reference Material/#Shi)].

In addition, among the above-mentioned IFO preserved strains, Ya36 is -m-
1 The American Type Culture Collection, a U.S. preservation institution
PE CULTURE COLLECTION (ATC
C) ) is a conserved strain of ( ATCC Catalog
ueofstrains I, /3 th Edit
ion, 197g (see needle review material)).

Hereinafter, for convenience, the microorganisms will be referred to as "KSY-!", etc.

In order to produce the β-hydroxy ester of the present invention, the above-mentioned microorganism is first inoculated into a suitable medium and cultured. Cultivation is carried out using normal yeast culturing methods, for example,
The cells are grown by shaking or stirring culture for a certain period of time in a medium containing potato, sucrose, glucose, casein decomposition products, etc.

Next, the above-mentioned starting material is added and further culturing is performed.

Further, after the bacterial cells have been grown, the bacterial cells are separated by an operation such as centrifugation, and the starting material is newly added to the bacterial cells together with the carbon source, water, etc., and the cells are further cultured. It's okay. The culture temperature is usually about 2g to 37C, preferably about 30C, and the culture time is usually about /2θ hours, preferably about 2 to 72 hours.

The obtained culture is filtered using, for example, Celite,
The p solution is extracted with an organic solvent in a conventional manner, then dried and the solvent is distilled off to obtain crude β-hydroxy ester (V). The purification method is, for example, as follows.

When the crude β-hydroxy ester (V) is directly subjected to silica column chromatography, the corresponding optical activity, t
s, n form [(A) or (B)] and Antl form [(
C) or (D)] can be separated. However, if it is difficult to separate and purify using the above method, the corresponding optically active Syn form [(A) or (B)] and Ant1 form [(C) or (D)] can be isolated by the following method. ] can be separated.

That is, the crude β-hydroxy ester (V) is
L end: lA/! ;f C7! ;ec4. mol) to/
Add 0% H (?4aC1, 20ml, stir and extrude filtrate at room temperature for S minutes, wash sequentially with water, special grade ethanol, and ether. After activating the generated zinc powder at 72θC for 70 minutes, anhydrous benzene;!rOvtl In addition, Methyl Cofflomo 7'a Bionate Milg, tAgli (1,2aq
, mol), coachofenaldehyde (1);
Add l/l, 7 sy and stir under reflux for 7 hours. After the reaction is completed, the mixture is acidified with 10% Hαaq and extracted from ether. The ether layer was saturated with NaHcO3aq, saturated Na
After successively washing with C1aq and dehydrating and drying with S04, the solvent was distilled off to obtain the crude product Methi7I/3-C2-chenyl)-3.
-Hydroxy-comethylzolopionate ((J')
+ (ri + (ff) + (ot)):g, '19g
gf (quantitative) is obtained. Since this was almost pure from TL=C and NMR, it was used in the next reaction without purification.

(3) + (to) + (4); IR (Cα4); 3SOOem (OH)/
'7.10cm-1, / 73! ;cm-”(sh)
(COOMe)NMR(A(7MHz FT% CD
Cl2)Syn; (3)+&')
ant I pieces + c Otsu) i233 d
J-'Z/ sec-Me l//b d J
-7,/sec-Me3.1s97s 3HCO
OMe 3.73! ; s 3HCOOMefu305t μ+OHfu029 dd /H 0H
J4.2 J number of units 9.27Sy
n (CJ)” (GQ) / antl (Ct)+(
ω) = //'739 (ratio according to COOMe) Reference example ((3) 10 cases + (8) + (Otsu))
C7) i-compound of β-hydroxyester (C7)
+ (g) + (a) + (4)):, 1-f is CH2Cl2
Dissolved in 5θ punishment, PDC; 29 y added room temperature test, day i push, 5to2 (c-100): SO of
The mixture was subjected to chromatography using Ac0Et to obtain an oily substance from the eluted portion of Ac0Et. This is repeated 5t02(C-2θO);2
Column chromatography using 0of, n-hexane/
β-ketoester (7);

(7); Original cumulative analysis (by Hlgh mass) cg
Calculated value as HIQO3S 19g, 03g Measured value 19g, 03/ IR (Cα4) 7670m (Calganiru) 1/7 Hiro0crn (ester) NMR (OtsuθMH2FT, cocg3, TMS)/,
5″/, 2 d J-Z/ 3Hsec-Me
3.706 s 3HC00Mθ442 Otsu2 q J=7/ /H Methine7A9'A d
d J8b=Left/J8o-1//HHa7/l
I'l dd Jba-&/ , Jbo-3,7/H
Hb7.7gS dd Jl) (=J, 7 J8o
-l/ /HHc [Yeast screening test] Yeast (KSY
-/ or KSY-39) was added, and after shaking culture at 30C for 2 days, about 5μ of compound (7) was added as a substrate.
Culture by shaking for an additional 3 days. The ridge was extracted with ether, and after distilling off the ether, the residue was analyzed by TLC (silica gel: n-hexane/Ac0Et-t//), 6 (7 MHz FT-
Searched by NMR.

As a result, at least KSY-G, 5% Otsu, Wa, 3%)
It was found that 2.161.24', 2 Otsu and 3 Otsu gave the product.

Example/ ('7) (4Q
(4) Add substrate (7)), 70θ■, washed bacterial cells (KSY-=7)/00F, sucrose/θ02.82401IθOM to a 2t flask, and stir at 30C for t hours. Add θ0t and wash C9H1° live bacterial cells (KSY-7430
01, Sucrose Calculated value = 1007, and cultured with shaking at 30C for a total of 4'5 hours Measured value: Yes. This reaction is repeated several times, and after the reaction is complete,
] and 1θ8 to extract liquid F from Ac0Et. Afti the IR (Cα4) AcOEt layer
After dehydration and drying with S04, the solvent is distilled off to obtain an oily mixture. Kore'x StO
2NMR (<zooMF (C-Cooo): column chromatography using 123g d, jl
-Hexane/Ac0E t=/9// ~9//
2. g9/ Reduced product from Cl-Cl elution part) +
(4); ! ; 3 g ni get. n-3,0
7! ;d from the elution part of hexane/Ac0E't-9// ant l <6); 3.700 sb
! ;get mi. The former further uses a Roper column.
! Purified with t30g t, 5ynu in order of bathing);
23 otl1v (yield/mu'7 1,9.t2 dd%
) ant+? 233mg (total 233+A
3=2qgmy &97dd (yield, 21/・%
). '7.21/l
Roper column (silica gel) Solvent: n-hexane/Ac0E t=3//Flow rate;
/Oml/mln [Physical properties of compound ('+) (5yn)] Elemental analysis (according to Hlgh Mass)々03S: OθO5l + o o, o s.

(C=fu/, CHα3, -27.IC) 3! ; 20
cm” (OH)1720cm-1%/73.!
;cm '(sh)' COOMeIzlFT-N
MR%CDC/3, TMSIJ='Z'/3Hse
c-Me J=7/ 4411 /H methine (J2,3
=44 Hiroshi) J=lAF /H0H 38COOMe J=taq, titt ++oH(J, 34
%) mixture of β-hydroxy esters (■+el)+(
3)+<1=)): ist, q, (+) MTPA (to
2. ? Pyridine θ in A”i (12θq,rnol)!
; Add tel and stir at room temperature for 3 days, then add water and extract from ether. The ether layer was washed with saturated Nactaq, dried over 4804 and evaporated to give an oil. Is this 5t02 (C-200)? 20?
Column chromatography using n-hexane/Ac0
From the elution part of E t-/q// (+) M T P A
Mixture of esters ((f) + (9) + Cl0) +
(//) ); Obtain Yugθ71Iv (yield g7%
).

σ) + rq> + (io)+ <ii>;40
0MHz FT-NMR (CDC/3, TMS) Syn
; (&) +(q) ant
l; (/(17)+(//)//ri9 d J
-'l/sec-Me iθ73 d J-7/
5ec-441/323 d J=2/sec
-Me i03'A d J-2/ sec-M [3,
6/ri s 3HCOOMe 3. Otsude 3
S 3HC00M13J', 57/s 3HC
OOMe, 1JO2s 3HCOOMe! ;20
d J-'Z/ Mu39hirod J=/θ7
MulI9'l d J=4:,? 13! ;
g d J-/θS 蓼例DA(2)R-H佼)R-H (,g) R-MTPA(+) ふ)R-MT
PA(+)I)(7)? /ll&q anhydrous:c-f
k j tnl Dissolved in VC, under argon stream, under OC stirring, added Zn(8H4)2/dry ether solution S-, stirred at same temperature for 7 hours, then added water, /θ%Hα
Acidify with water and extract from ether. The ether layer was sequentially washed with saturated NaHCO3aq and saturated NaαaQ,
After dehydration and drying with Mpso4, the solvent was distilled off to obtain an oily substance: /4t
Get Otq.

■+(Q:(+θθMHz FT-NMRCDCts
, TMS) 32 d J-7, 3sec-M
e2ggOqq, J-1A4'l/
/H methine 3.2/3 1:+r,s
/HOH3, Ag! ;s,? H.C.O.O.O.
Mec f a n t l (G)+(/;)):
COOMe:C3,73011) The above Syn body ((
3)+(4')): 27 wtl(+)MTPA
α'I/x9 (i 2 eq, mol) was dissolved in pyridine 0. ．． 5-m7! After stirring at room temperature for 6 hours, add water and extract from ether. Saturate the ether layer with NaCtaq.

After washing with water and dehydrating and drying with A4 SO, the solvent was distilled off to obtain an oil.・Prepare this using a silica glu plate TLc (,, -hexane/Ac0E t=3//
), (+) M T P A ester (Syn form): (1) + Cq; 3 g,! Obtain r~ (yield qchi).

(lr)+(q): (4'0(7MHz FT NM
R, cocg3, TMS) i/gOd J-7,/
seC-Mei32'l d J-A,g
sec-Me3, so7 d J-0,9ON
θ 3, 'l'l-9tj J-C90Me3, Otsu/g
s 3rd COOMe3,! r73
S,? HCOOMemu379 (I J='
2/ Haya+OM T P A(+)mu1Iq3 d
J=g, 3 H+OMTPA←)(s)+(9): Elemental analysis (by Hlgh Mass) C19H1905
Calculated value as F3S Da/muOqO Measured value Hiro/mu09θ Reference example 3 (a) C) Compound (p) obtained in Example 1: , t , 5-1
19, ←) MTPA&:za3 '12 (l!eq, m
Add pyridine θ3 wtl to 737
After stirring for an hour, add water and extract from ether. The ether layer was washed with saturated Naαaq, dehydrated and dried over h1gS04, and the solvent was distilled off to obtain an oil. This was prepared using a silica gel plate for TLC (solvent: n-hexane/Ac
0Et-3// ), (+) M T P A
Ester (9); g 3 wq is obtained.

(9):'+00MH2FT NMR, (CDCj3
, TMS) 1323 d J-'7. / se
c-Me3.1. /II s 3HCOOMe C
? 'l/9)mu320dJd'7. / cf (//); 1179 d J-Muza optical purity: ? +/q+7A/ Reference example 6 0MTPbe+)3). H2N, OMTPK+) (q)
C/2) (9):
73 mII was dissolved in CH2 (J2 Otsu-), and ozone was blown into the solution for 30 minutes while cooling in a dry bath with acetone.

After that, return to room temperature and add 30% H2O2aq, 2
ml and after stirring for 20 minutes, water is added and extracted from ether. Wash with ethereal saturated Naαaq, Mg
After dehydration and drying with SO4, the solvent was distilled off to obtain an oily substance. After treatment with a CH2N2/ether solution, the methyl post-mortem solvent is distilled off to give an oil. This was prepared using a silica gel plate for TLC (solvent: n-hexane/Ac0Et
-J//) and dimethyl ester (9IIv
I can get it.

(/, 2)? Hiro θ (7MHz FT NMR (C
DC (3, TMS) cf antlpocle 12/! ; d J-7, 3sec-Me 117
3 d J=73 sec-Me left 793 d J
-,? ,+Re÷OMTPbe+) Reference Example 7 0H6MTPK+) (B)(//) Compound (A) obtained in Example/: s II vq
, (+)MrpAa;g 3 rxl (12eq
, mol) was added with 0.3 rttl of viridi, and after stirring at room temperature for 7 hours, water was added and extracted from ether. Wash the ether layer with saturated Nactaq, A! 4
After dehydration and drying with So4, the solvent is distilled off to obtain an oil. This was prepared using a silica gel plate for TLC (solvent; n
-hexane/ Ac0At -3// ), (+
) M T P A /ester (//) /θyu5~
get.

(//) ;4'00MHz FT NMR (CDcg
3, TMS) i073 d J-'13 se
c-Me cf (/θ]; Reference example g C1/) C/3) C//
):92! Dissolve q in CH2 (j2 b Ml; and blow in ozone for 30 minutes while cooling with dry ice-acetone. Then return to room temperature and add 30% H2O2aQ-5r.
After stirring for 20 minutes, water is added and extracted from ether. The ether layer was washed with saturated Naαaq, Mg
After dehydration and drying with SO4, the solvent is distilled off to obtain an oil. After treating this with CH2N2/ether solution to form a methyl layer,
Distillation of the solvent yields an oil. This was prepared by TLC using a silica dull plate (solvent; jl-hexay'
/Ac0Et-3//n to give dimethyl ester C/3); 3.3 WIg.

(/3); Hiro (70MHz FT-NMR (CDα3
, TMS) cf (AntlpOdθ) 0.67 d J-1sec-Me II'72
d J-2/ sec-Me Examples a to g Mw4/6 (C1gH19SO5F31 (1)
(10) KSY-G, S, who obtained good results in the primary screening
17, Za, /6.2 Otsu, 36 was considered. A small amount of yeast scraped from the slant was added to the liquid medium ioomi, and after shaking culture at 3θC for 3 days, about 3 Oyn of substrate (7) was added.
g and cultured with shaking for an additional 3 days. Each reaction is then filtered through Celite and the filtrate is extracted from ether. After dehydrating and drying the ether layer with 4SO4, the solvent was distilled off to obtain each oil.

(+) MTPAO7A W (12e
After adding pyridine θ5 and stirring at room temperature for 73 hours, water was added and extracted from ether. Wash with ethereal saturated Naclaq, A4js
After dehydration and drying at 4 o'clock, the solvent was distilled off to obtain an oily product.

This was subjected to preparative TLC (solvent di-n-hexane/Ac0Et-3//) using a silica dull plate, and (
+) MTP A ester obtained.

The results are shown in the following formula and Table 2.

6FA- too Old banquet)) Rei fishing brown sa Q song Continuation of page 1 0 shots clear person Hiroko Koshichi 2-1 Hirosawa, Wako City Physical and Chemistry Research Inside the office 0 shots Akiya Furuichi 4-18-7 Nishiota, Tama-ku, Kawasaki City 401

Claims (1)

[Claims] (1) General formula: (wherein, X is α-C) (3 or β-CH3
, Y represents α-OH or β-OH. ) β-hydroxy ester represented by: @ The compound according to claim 1, wherein X is α-CH3 and Y is α-OH. G3) The compound according to claim 1, wherein X is α-CH3 and Y is β-OH. 2) The compound according to claim 1, wherein X is β-CH3 and Y is α-OH. (To) The compound according to claim 1, wherein x is β-CH3 and Y is β-OH. (6) General formula: (In the formula, R represents an alkyl group.) A compound represented by Candida (CandLda)
Genus, Endomycopsls
Genus Hansenula, Genus Kloeckera, Genus Lipomyces (Llp
saccharomyces), saccharomyces
omyces), genus Schizosatsuka CI”f Ises (Sc
A compound represented by the general formula: (wherein R is the same as above) is contacted with a microorganism selected from the microorganism group belonging to the genus hlzosaccharomyces and genus Sporobolomyces, or its bacterial cells. A method for producing a β-hydroxy ester, characterized in that it obtains a β-hydroxy ester.